The present invention relates to novel urokinase production inhibitors, angiogenesis inhibitors and a preventive or therapeutic method with use of the inhibitor.
Urokinase (urine-type plasminogen activator, i.e., uPA) is a protease which is highly specific to a single peptide linkage in plasminogen. Urokinase converts plasminogen to plasmin which is an active fibrinolytic enzyme. Activated plasmin acts on fibrin, fibronectin and laminin, converts inactive matrix metalloprotease (MMP) to active MMP and promotes liquefaction of collagen, a component of the basement membrane. In addition to the function of a protease, urokinase also has activity to promote the migration of vascular endothelial cells and to promote tube formation on Matrigel, serving an important function in angiogenesis.
The physiological processes in which urokinase participates include, for example, angiogenesis, osteoanagenesis, nidation, infiltration of immunocytes into inflammatory sites, ovulation, spermatogenesis, anagenesis for the repair of wounds and differentiation of organs, local infiltration of fibrosis and tumor into adjacent regions, metastatic spread of tumor cells from the primary site to a secondary site, and disorganization in arthritis. Accordingly urokinase inhibitors have activities against angiogenesis, arthritis, inflammation, invasion, metastasis, osteoporosis and retinopathy (angiogenesis-dependent retinopathy), contraceptive activity and activity to inhibit growth of tumor. It is therefore expected to develop medicinals which will act on urokinase as a molecular target. Reports are made on useful effects of anti-urokinase monoclonal antibodies and some urokinase inhibitors. For example, it is reported that anti-urokinase monoclonal antibodies block the invasiveness of tumor cells in vitro [Cancer Res., 51, 3690-3695(1991); Exp. Cell Res., 192, 453-459(1991)]. Reportedly, amiloride, a known urokinase inhibitor having medium efficacy, blocks the metastasis of tumors in vivo [Anticancer Res., 8, 1373-1376(1988)], and prevents angiogenesis or development of capillary reticular structures in vitro [J. Cell Biol. 115(3 Pt 2):402a(1991)].
The ozonide derivatives according to the present invention include some compounds which are known [for example, J. Am. Chem. Soc. (1984), 106(10), 2932-6, J. Org. Chem. (1985), 50(9), 1504-9, J. Org. Chem. (1990), 55(13), 4221-2, Ann. Chim. (Paris) 9 (7-8), 359-97(1964), J. Am. Chem. Soc. (1983), 105(8), 2414-26, J. Org. Chem. (1993), 58(1), 135-41, J. Org. Chem. (1985), 50(2), 275-7, etc.]. However, the literature relates only to preparation processes and optochemical properties, and nothing has been known about the fact that these compounds have high activity to inhibit production of urokinase and are useful as urokinase production inhibitors and angiogenesis inhibitors.
An object of the present invention is to provide novel urokinase production inhibitors and angiogenesis inhibitors, and a preventive or therapeutic method with use of such an inhibitor.
The present invention provides a urokinase production inhibitor comprising as an active component an ozonide derivative represented by the formula (1) 
wherein A is an oxygen atom or Nxe2x80x94R (wherein R is phenyl or phenyl having as a substituent lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom); B is an oxo group or xe2x80x94R4; and (1) when A is an oxygen atom, R1 is a hydrogen atom, lower alkyl having 1 to 6 carbon atoms, phenyl or phenyl having as a substituent lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms, lower alkoxycarbonyl having 2 to 7 carbon atoms or a halogen atom, R2 is phenyl or phenyl having as a substituent lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom, R3 is a hydrogen atom, lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom, B is an oxo group or xe2x80x94R4, R4 is a hydrogen atom, lower alkyl having 1 to 6 carbon atoms, a halogen atom, lower alkanoyl having 2 to 6 carbon atoms or phenyl, and R5 is a hydrogen atom, lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom or forms an aromatic 6-membered ring when combined with R4; or (2) when A is Nxe2x80x94R, R1 is a hydrogen atom, lower alkyl having 1 to 6 carbon atoms, phenyl or phenyl having as a substituent lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom, R2 is a hydrogen atom or lower alkyl having 1 to 6 carbon atoms, R3 is a hydrogen atom, lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom, B is xe2x80x94R4, R4 is a hydrogen atom, phenyl or phenyl having as a substituent lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom, and R5 is a hydrogen atom, lower alkyl having 1 to 6 carbon atoms, lower alkoxyl having 1 to 6 carbon atoms or a halogen atom or forms an aromatic 6-membered ring when combined with R4.
The invention further provides a urokinase production inhibitor composition containing an ozonide derivative represented by the formula (1) and a pharmacologically acceptable carrier.
The invention further proposes use of an ozonide derivative represented by the formula (1) for preparing a urokinase production inhibitor.
The invention further provides a method of inhibiting production of urokinase by administering to a human an effective amount of an ozonide derivative represented by the formula (1).
The invention further provides an angiogenesis inhibitor comprising as an active component an ozonide derivative represented by the formula (1).
The invention further provides an angiogenesis inhibitor composition containing an ozonide derivative represented by the formula (1) and a pharmacologically acceptable carrier.
The invention further proposes use of an ozonide derivative represented by the formula (1) for preparing an angiogenesis inhibitor.
The invention further provides a method of preventing or treating a disease accompanying angiogenesis by administering to a human an effective amount of an ozonide derivative represented by the formula (1).
The invention further provides an agent for preventing or treating a malignant tumor comprising as an active component an ozonide derivative represented by the formula (1).
The invention further provides a composition for preventing or treating a malignant tumor containing an ozonide derivative represented by the formula (1) and a pharmacologically acceptable carrier.
The invention further proposes use of an ozonide derivative represented by the formula (1) for preparing an agent for preventing or treating a malignant tumor.
The invention further provides a method of preventing or treating a malignant tumor by administering to a human an effective amount of an ozonide derivative represented by the formula (1).
The invention further provides an agent for preventing or treating metastasis of a tumor comprising as an active component an ozonide derivative represented by the formula (1).
The invention further provides a composition for preventing or treating metastasis of a tumor containing an ozonide derivative represented by the formula (1) and a pharmacologically acceptable carrier.
The invention further proposes use of an ozonide derivative represented by the formula (1) for preparing an agent for preventing or treating metastasis of a tumor.
The invention further provides a method of preventing or treating metastasis of a tumor by administering to a human an effective amount of an ozonide derivative represented by the formula (1).
The compounds serving as the active component of the invention and represented by the formula (1) have the following basic structures (A) to (D). 
According to the invention, the group B included in the formula (1) is an oxo group or a group represented by xe2x80x94R4, and the group B is combined with a ring by a single bond or double bond. When B is the group xe2x80x94R4, this group can be combined with R5 to form an aromatic 6-membered ring.
According to the invention, examples of lower alkyl groups having 1 to 6 carbon atoms are straight-chain or branched lower alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl, among which preferable are those having 1 to 4 carbon atoms, i.e., methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl.
Examples of lower alkoxyl groups having 1 to 6 carbon atoms are straight-chain or branched lower alkoxyl groups having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butyloxy, sec-butyloxy, tert-butyloxy, pentyloxy and hexyloxy, among which methoxy and ethoxy are preferable.
Examples of halogen atoms are fluorine atom, chlorine atom, bromine atom and iodine atom.
Examples of lower alkoxycarbonyl groups having 2 to 7 carbon atoms are straight-chain or branched lower alkoxycarbonyl groups having 2 to 7 carbon atoms, such as methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, i-propoxycarbonyl, n-butyloxycarbonyl, sec-butyloxycarbonyl, tert-butyloxycarbonyl, pentyloxycarbonyl and hexyloxycarbonyl, among which methoxycarbonyl and ethoxycarbonyl are preferable.
Examples of lower alkanoyl groups having 2 to 6 carbon atoms are alkanoyl groups having 2 to 6 atoms, such as acetyl, propionyl, butyryl, pentanoyl and hexanoyl, among which acetyl is preferable.
Preferable among the active components represented by the formula (1) of the invention are as follows.
[1] When having the basic structure (A): Preferable are compounds wherein A is an oxygen atom, R1 is a hydrogen atom, methyl, ethyl, phenyl or phenyl having as a substituent methyl, methoxy, methoxycarbonyl or a chlorine atom, R2 is phenyl or phenyl having as a substituent methyl, methoxy or a chlorine atom, R3 is a hydrogen atom, B is oxo group or xe2x80x94R4, R4 is a hydrogen atom, methyl, ethyl, isopropyl, tert-butyl, a chlorine atom, acetyl or phenyl, and R5 is a hydrogen atom. More preferable are compounds wherein A is an oxygen atom, R1 is a hydrogen atom, methyl or phenyl, R2 is phenyl or phenyl having methoxy as a substituent, R3 is a hydrogen atom, R4 is a hydrogen atom, methyl, isopropyl or phenyl, and R5 is a hydrogen atom.
[2] When having the basic structure (B): Preferable are compounds wherein A is an oxygen atom, R1 is a hydrogen atom or phenyl, R2 is phenyl, R3 is a hydrogen atom, and R5 is combined with R4 to form a phenyl ring.
[3] When having the basic structure (C): Preferable are compounds wherein A is Nxe2x80x94R, R is phenyl, R1 is a hydrogen atom or phenyl, R2 is a hydrogen atom, R3 is a hydrogen atom, R4 is a hydrogen atom or phenyl, and R5 is a hydrogen atom.
[4] When having the basic structure (D): Preferable are compounds wherein A is Nxe2x80x94R, R is phenyl, R1 is a hydrogen atom or phenyl, R2 is a hydrogen atom, R3 is a hydrogen atom, and R5 is combined with R4, forming a phenyl ring.
Due to the presence of asymmetric carbon, optical isomers are present for the compounds represented by the formula (1) and useful as the active components of the invention. The isomers of the respective compounds and mixtures of such isomers are also included in the active components of the invention. Exo-isomers and endo-isomers are also present due to different configurations. While the active components of the invention include these isomers and mixtures thereof, preferable are exo-isomers.
The compounds represented by the formula (1) and serving as the active components of the invention are prepared by substantially the same processes as disclosed, for example, in J. Am. Chem. Soc. (1984), 106(10), 2932-6, J. Org. Chem. (1985), 50(9), 1504-9, J. Org. Chem. (1990), 55(13), 4221-2, Ann. Chim. (Paris) 9 (7-8), 359-97(1964), J. Am. Chem. Soc. (1983), 105(8), 2414-26, J. Org. Chem. (1993), 58(1), 135-41, and J. Org. Chem. (1985), 50(2), 275-7. More specifically, these processes are as follows.
When A is an oxygen atom, compounds represented by the formula (1a) and serving as the active component of the invention are prepared by the following reaction step i. 
wherein R1 to R3, B and R5 are same as above.
[Reaction Step i]
A compound represented by the formula (2) is reacted with ozone in a suitable solvent, whereby a compound represented by the formula (1a) is obtained. The solvent is not limited specifically insofar as the solvent does not participate in the reaction. Examples of useful solvents are methylene chloride, chloroform, carbon tetrachloride, ether, tetrahydrofuran, benzene and petroleum ether. For the reaction, ozone is used in 0.5 to 5 moles, preferably 1 to 3 moles, per mole of the compound (2). The reaction is conducted usually at a temperature of xe2x88x9210 to 20xc2x0 C., preferably xe2x88x925 to 5xc2x0 C., for 5 to 30 minutes, preferably for 10 to 15 minutes.
The compound represented by the formula (2) is a known compound disclosed in J. Org. Chem., 6, 534(1941), J. Am. Chem. Soc., 56, 1337(1934), ibid., 65, 567(1943), ibid., 106, 2932(1984), Acta Chem. Scand., Ser. B. B28, 295(1974), Helv. Chim. Acta, 30, 1320(1947), etc., or prepared by substantially the same process as disclosed in the literature.
(2) When A is Nxe2x80x94R, compounds represented by the formula (1b) and serving as the active component of the invention are repared by the following reaction step ii. 
wherein R, R1 to R5 are same as above.
[Reaction Step ii]
A compound represented by the formula (3) is reacted with a compound represented by the formula (4) in a suitable solvent, whereby a compound represented by the formula (1b) is obtained. The solvent is not limited specifically insofar as the solvent does not participate in the reaction. Examples of useful solvents are methanol, ethanol, benzene, ether, tetrahydrofuran, dioxane, methylene chloride, chloroform and carbon tetrachloride. For the reaction, the compound (4) is used in 0.5 to 5 moles, preferably 1 to 3 moles, per mole of the compound (3). The reaction is conducted usually at a temperature of 0 to 50xc2x0 C., preferably 10 to 30xc2x0 C., for 1 to 48 hours, preferably for 10 to 24 hours.
Incidentally, the compound represented by the formula (3) can be prepared from the compound represented by the formula (1a) and obtained by the reaction step i, by reducing the compound (1a) with a reducing agent in a suitable solvent. The solvent is not limited specifically insofar as the solvent does not participate in the reaction. Examples of useful solvents are benzene, ether, methylene chloride, chloroform and petroleum ether. Examples of reducing agents usable are triphenylphosphine, trimethyl phosphite, triphenyl phosphite and dimethyl sulfide, among which triphenylphosphine is preferable. For the reaction, the reducing agent is used in 0.5 to 5 moles, preferably 1 to 3 moles, per mole of the compound (1a) . The reaction is conducted usually at a temperature of 0 to 50xc2x0 C., preferably 10 to 30xc2x0 C., for 1 to 48 hours, preferably for 10 to 24 hours.
The compound (1a) or (1b) obtained by the foregoing reaction step can be easily isolated from the reaction mixture and purified by a usual separation or purifying method such as column chromatography, recrystallization or vacuum distillation.
The active component of the present invention is used usually in the form of usual pharmaceutical preparations. Such preparations are formulated using diluents or excipients which are usually used, such as fillers, extenders, binders, humectants, disintegrators, surfactants and glazing agents. The pharmaceutical preparations can be in various forms each as selected in conformity with the therapeutic purpose. Examples of typical forms are tablets, pills, powders, liquid preparations, suspensions, emulsions, granules, encapulated preparations, suppositories, injections (liquid, suspension, etc.), ointments, etc.
Examples of carriers for use in preparing tablets are excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose and silicic acid, binders such as water, ethanol, propanol, syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and polyvinylpyrrolidone, disintegrators such as dried starch, sodium alginate, agar powder, laminaria powder, sodium hydrogencarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium laurylsulfate, stearic acid monoglyceride, starch and lactose, disintegration suppressants such as sucrose, stearic acid, cacao butter and hydrogenated oils, absorption promoters such as quaternary ammonium bases and sodium laurylsulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, glazing agents such as purified talc, stearic acid salts, boric acid powder and polyethylene glycol, etc. When required, the tablets can be those having a usual coating, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double-layer tablets and multi-layer tablets.
Examples of carriers for use in preparing pills are excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oils, kaolin and talc, binders such as gum arabic powder, tragacanth powder, gelatin and ethanol, disintegrators such as laminaria and agar, etc. Examples of carriers for use in preparing suppositories are polyethylene glycol, cacao butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides, etc. Encapsulated preparations are obtained in the usual manner by mixing the compound of the invention with such carriers as exemplified above, and filling the mixture into hard gelatin capsules or soft capsules.
When to be formulated into injections, liquid preparations or suspensions are sterilized and preferably made isotonic with blood. Examples of diluents for use in preparing such preparations are water, aqueous solution of lactic acid, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc. The pharmaceutical preparations may have further incorporated therein sodium chloride, glucose or glycerin in an amount sufficient to prepare an isotonic solution. The solution may also contain a usual auxiliary solubilizing agent, buffer, analgesic or the like added thereto. Further when required, the pharmaceutical preparations may have incorporated therein a coloring agent, preservative, perfume, flavoring, sweetener and the like, and other medicinals. Examples of diluents usable for formulating preparations in the form of a paste, cream or gel are white petrolatum, paraffin, glycerin, cellulose derivatives, polyethylene glycol, silicon and bentonite. The amount of the compound of the invention represented by the formula (1) or a salt thereof (active component compound) to be incorporated in the foregoing pharmaceutical preparations is not limited specifically but suitably determined from a wide range. It is usually desirable that the pharmaceutical preparations contain 1 to 70 wt. % of the active component.
The method of administering the pharmaceutical preparation of the invention is not limited specifically but determined according to the form of preparation, age, sex and other conditions of the patient and degree of symptom of the patient. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are given orally. Injections are intravenously given singly or as mixed with a usual auxiliary solution such as glucose or amino acid solution. When required, the injection is singly administered intramuscularly, intracutaneously, subcutaneously or intraperitoneally. Suppositories are introduced into the rectum.
The dosage of the active component of the pharmaceutical preparation of the invention is suitably determined according to the mode of administration, age, sex, other conditions and degree of symptom of the patient. The compound of the invention serving as the active component is administered usually at a daily dose of about 1 to about 1000 mg/kg body weight, preferably about 10 to about 100 mg/kg body weight. The preparation can be given once or dividedly up to four times per day.
The medicinal of the invention has high activity to inhibit production of urokinase and is useful as a urokinase production inhibitor.
The medicinal of the invention also has high angiogenesis inhibitory activity and is therefore useful as an agent for treating or preventing diseases accompanying angiogenesis. The diseases accompanying angiogenesis include, for example, malignant tumors, metastases of tumors, benign tumors (e.g., hemangioma, acoustic neurinoma, neurofibroma, trachoma and pyogenic granuloma), vascular dysfunction, inflammation and immune disorders, Behcet""s syndrome. gout, arthritis, chronic articular rheumatism, psoriasis, diabetic retinopathy and other diseases originating in ocular blood vessels (e.g., fibroplasia of the posterior lens, macular degeneration, rejection of corneal transplant and angiogenesis glaucoma), osteoporosis, etc. By virtue of the above activity, the medicinal of the invention especially has high activity to inhibit the growth of tumors and to inhibit the metastasis of tumors and is therefore useful as an agent for preventing or treating malignant tumors and as an agent for preventing or treating the metastasis of tumors. Although not limited particularly, examples of malignant tumors are cancer of the head and neck, esophageal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, testicular tumor, bone and soft part sarcomas, cervical cancer, skin cancer, brain tumor and like solid malignant tumors. Although not limited specifically, metastases of tumors are those of tumors which are sensitive to the therapy of the invention, including, for example, metastases to the liver, lungs and other organs, and metastases to lymph nodes, bones, the brain and other tissues or structures.